The Journal of Neuroscience, December 1990, IO(1‘2): 37934900
Dynorphin A-( l-l 7) Induces Alterations in Free Fatty Acids, Excitatory Amino Acids, and Motor Function Through An Opiate- Receptor-Mediated Mechanism
Rohit Bakshi,’ Amy H. Newman,2 and Alan I. Faden’ ‘Center for Neural Injury, Department of Neurology, University of California, San Francisco, California 94121 and Neurology Service (127), Department of Veterans Affairs, San Francisco, California 94121, and *Department of Applied Biochemistry, Walter Reed Army Institute of Research, Washington, DC. 20307-5100
The endogenous opioid dynorphin A-( l-1 7) (Dyn A) has been Phamacological studies with opioid-receptor antagonists sup- implicated as a mediator of tissue damage after traumatic port the concept that endogenous opioids contribute to the spinal cord injury (TSCI) and causes hindlimb paralysis when pathophysiology of tissue damageafter CNS trauma (Faden et administered intrathecally. Motor impairment following in- al., 1981; Flamm et al., 1982; Hayes et al., 1983; Arias, 1985; trathecal Dyn A is attenuated by antagonists of excitatory Inoue, 1986; McIntosh et al., 1987). Dynorphin A (Dyn A), a amino acids (EAAs); whether opioid receptors mediate such 17-amino acid opioid peptide thought to be an endogenous injury has been questioned. TSCI causes various biochem- ligand for the K-Opiate receptor (Chavkin and Goldstein, 1981; ical changes associated with secondary tissue damage, in- Yoshimura et al., 1982), has been implicated as a secondary cluding alterations in tissue amino acids, phospholipids, and injury factor after spinal cord (Faden et al., 1985) and brain fatty acids. Such changes reflect injury severity and corre- trauma (McIntosh et al., 1987). Following traumatic spinal cord late with motor dysfunction. The present studies examined injury (TSCI), levels of dynorphinlike immunoreactivity in- whether dynorphin administration causes similar biochemi- creasein proportion to the degreeoftrauma (Faden et al., 1985), cal alterations and whether effects of Dyn A can be modified whereastreatment with dynorphin antiserum or K-selectiveopi- by treatment with opioid-receptor antagonists. At 24 hr after ate-receptor antagonistsimprove neurological recovery (Faden, intrathecal Dyn A, there were significant declines in tissue 1990). levels of glutamate, aspartate, and glycine. Increases in total Consistentwith its putative pathophysiologic role, Dyn A and free fatty acids were found at 2 and 24 hr, reflecting changes related fragments,administered intrathecally, causechronic pa- in both saturated and unsaturated components, which were ralysis (Faden and Jacobs, 1984; Stevensand Yaksh, 1986), loss associated with significant decreases in tissue cholesterol of the tail-flick reflex (Herman and Goldstein, 1985), neuroan- and phospholipid phosphorus at the earlier time point. Each atomical damage(Caudle and Isaac, 1987; Long et al., 1988), of these neurochemical changes, as well as corresponding and a decreasein local spinal cord blood flow (Long et al., 1987; motor deficits, were limited by pretreatment with the opioid Thornhill et al., 1989). Effects of dynorphin on the tail-flick antagonist nalmefene. In separate experiments, both nal- reflex or motor function are prevented by treatment with NMDA mefene and the selective K-opioid antagonist nor-binaltor- antagonists(Caudle and Isaac, 1988; Long et al., 1989a; Bakshi phimine (nor-BNI) limited dynorphin-induced motor dysfunc- and Faden, 1990a,b), suggestingthat dynorphin-induced neu- tion; effects of nor-BNI were dose related, and those of rological dysfunction involves the releaseof excitatory amino nalmefene were stereospecific. Therefore, behavioral and acids (EAAs). However, the role of opiate receptors has been neurochemical consequences of Dyn A administration are more controversial. Some groups have shown that opiate-re- mediated in part through opiate receptors, most likely K-e- ceptor antagonistsattenuate Dyn A-induced paralysis (Przew- ceptors. These studies indicate that phospholipid hydrolysis locki et al., 1983; Spampinato and Candeletti, 1985; Faden, and release of EAAs may contribute to dynorphin-induced 1990), whereasothers have not (Herman and Goldstein, 1985; tissue damage, suggesting for the first time a potential link- Stevensand Yaksh, 1986; Longet al., 1988,1989b). In addition, age among opioid, excitotoxin, and membrane lipid mech- nonopioid fragments of Dyn A, including Dyn A-(2-1 7) and anisms of secondary injury after neurotrauma. Dyn A-(3- 13), also causeparalysis, though with markedly less potency than Dyn A-( 1- 17) (Faden and Jacobs, 1984; Stevens and Yaksh, 1986). A recent report critically reviews this con- Received Mar. 12, 1990; revised June 21, 1990; accepted July 24, 1990. troversy and provides experimental evidence that both opioid This work was supported by NIH Grant ROl NS23422. We wish to thank and nonopioid mechanismsplay a role in Dyn A-induced pa- Florence Cheng, Brendan Chan, and Vicky Cardenas for technical assistance and Dr. Steven Graham for reviewing this work. We adhered to the principles enu- ralysis (Faden, 1990). merated in the Guide for the Care and Use of Laboratory Animals, prepared by TSCI causesincreased tissue levels of free fatty acids (FFAs; the Committee on Care and Use of Laboratory Animals of the Institute of Lab- Demediuk et al., 1985; Faden et al., 1987) and decreasedtissue oratory Resources, National Research Council [DHEW Pub. No. (NIH) 85-23, 19851. R.B. is a recipient of an Alpha Omega Alpha Research Scholarship to levels of EAAs (Demediuk et al., 1989). Releaseof FFAs after Medical Students. trauma reflects phospholipid hydrolysis and may contribute to Correspondence should be addressed to Alan I. Faden, M.D., Neurology Service (127) Department of Veterans Affairs, 4 150 Clement Street, San Francisco, CA subsequenttissue damage either through direct toxic effects(Chan 94121. et al., 1983) or through the actions of such metabolic products Copyright 0 1990 Society for Neuroscience 0270-6474/90/123793-08$03.00/O as thromboxanes (Hsu et al., 1985). Early releaseof EAAs into 3794 Bakshi et al. * Dynorphan-Induced Neurochemical Changes the extracellular space after spinal cord trauma (Panter et al., ing with a solution of 2-p-toluidinyl napthalene-6-sulfonate (Jones et 1990) is believed to lead to subsequent loss of total tissue levels al., 1982). Lipid bands were scraped from the plates prior to analysis. Cholesterol, in the presence of silica gel, was quantitated by the method (Demediuk et al., 1989). Pharmacological studies indicate that of Bowman and Wolf (1962). FFAs were extracted from the silica gel these EAA changes contribute to delayed tissue damage after using chloroform : methanol (2: 1, vol/vol). Silica gel was removed by spinal cord trauma (Faden and Simon, 1988) or brain trauma filtering through 0.2-pm nylon filters into conical centrifuge tubes. A (Hayes et al., 1988; Faden et al., 1989). The present experiments heptadecanoate internal standard was added, and the filtrate was evap- were intended to explore whether changes in FFAs and EAAs orated under N,. Fatty acid methyl esters (FAMES) were prepared using a modification of the method of Allen et al. (1984). To each centrifuge in Dyn A-induced injury parallel those after traumatic injury tube, 20 ~1 0.5 M NaOH, 80 J N,N-dimethyl acetamide, and 40 J and whether such changes are opioid-receptor mediated. methyl iodide was added, with vortexing after every addition. The re- action mixtures were then heated at 65°C for 10 min and allowed to Materials and Methods cool. Ninety microliters pyridine was added with vortexing, and the Intrathecal infusion model. Male Sprague-Dawley rats, weighing 300- tubes were again heated at 65°C for 10 min. After cooling, 0.8 ml 0.1 350 gm, were anesthetized with sodium pentobarbital(70 mg/kg, i.p.). M phosphoric acid equilibrated with ethylene chloride was added, fol- An intrathecal line was implanted to the eighth thoracic vertebral level lowed by 25 ~1 ethylene chloride. The tubes were vortexed for 30 set using a modification of the method of Yaksh and Rudy (1976), as and centrifuged at 500 x g for 2 min. Two microliters of the ethylene previously detailed (Bakshi and Faden, 1990a). Briefly, polyethylene chloride lower phase was removed with a Hamilton syringe and injected tubing (PE- 10) was implanted into the subarachnoid space through the into a Perkin-Elmer Sigma 300 gas chromatograph for separation and atlanto-occipital membrane and passed to T8. The catheter was then auantitation of FAMES. A Sunelco (Bellefonte. PA) nrenacked 5% DEGS- secured below the skin and the wound sutured. Animals were allowed PS (Supelcoport 100/200 mesh) column was ‘used wi
TOTAL FFA’s a 12 in 1 16:O T 35 a a 30 T
25
.G 2P 20 veh dyn ml a a F \ 15 B 9 - 18:O
10 2
T 5 1
0 0 veh dyn nal veh ml veh dyn ml veh dw nal dyn
2h 24 h Figure 1. Changesin spinalcord FFAs (expressedas pg/mg protein) a at infusionsite after intrathecal administration ofvehicle [saline + saline 20:5 (veh)],saline + 24 nmolDyn A (dyn), or 8 nmol nalmefene+ Dyn A (nal)at 2 and24 hr postinfusion.Values are expressed as means f SEM (n = 5-7 pergroup). a, differentfrom veh (p < 0.05);*, differentfrom dyn (p < 0.05).
veh dyn nal veh dyn nal Figure 3. Significantchanges in individual FFAs asin Figure1 at 24 hr postinfusion.a, differentfrom veh (p < 0.05);au, differentfrom veh 0, < 0.005);*, differentfrom dyn @ < 0.05).
veh dyn lid Amino acids 4, 51 18:O a 18:i ; Twenty-four hr after infusion, Dyn A-( I- 17) causedsignificant d I decreasesin total tissue levels of aspartate,glutamate, and gly- tine (Fig. 5), but only modest decreasesin serine, GABA, glu- tamine, alanine, and taurine (data not shown). The changesin aspartate,glutamate, and glycine were limited by pretreatment with nalmefene(Fig. 5). 1
- 0 ELI Neurologic outcome veh dyn veh dyn In thesesame rats, nalmefenetreatment significantly improved Dyn A-induced mortality and paralysis (measuredby neuro- scoreand angle-boardscore) as compared to vehicle-pretreated 9 18:3 2] 22:6 controls (eachp < 0.05; Table 1). 1 i 6 Study 2 1 Efects of nalmefene, nor-BNI, or vehicle alone
3 Infusion of saline, (+)- or (-)nalmefene, or 20 nmol nor-BNI had no detectableeffect on hindlimb function; however, 35 and 50 nmol nor-BNI given alone causedmild motor deficits that resolved by 10 min (35 nmol) or 30 min (50 nmol; data not veh Ml veh nal dyn dyn shown).These transient effectsare similar to observationsmade Figure 2. Significantchanges in individual FFAs asin Figure 1 at 2 hr postinfusion.a, differentfrom veh (p < 0.05);*, differentfrom dyn with competitive NMDA antagonists(Caudle and Isaac, 1988; 0, i 0.05). Bakshi and Faden, 199Oa). 3796 Bakshi et al. l Dynorphan-Induced Neurochemical Changes
6 *
5
4
veh dyn veh dv 2h 2h
2.5,
2.0
1.5 Figure 4. Corresponding changes in spinal cord cholesterol and total phos- 1.0 pholipid phosphorus (Zipidphos) of an- imals from Figures l-3 at the infusion 0.5 site after intrathecal administration of vehicle [saline + saline (veh)], saline + 0.0 Dyn A (dyn), or nalmefene + Dyn A veh dw llal veh dyn InI (nal) at 2 and 24 hr postinfusion. Each mean and SEM is expressed as PmoV 24 h 24 h mg protein. a, different from veh @ < 0.05); *, different from dyn (p < 0.05). Cholesterol H Lipid Phos
Blockade of Dyn A-induced efects also significantly improved other behavioral measuresof re- Intrathecal infusion of 24 nmol Dyn A produced flaccid hind- covery (Fig. 7). Additionally, nalmefene(stereospecifically) and limb weaknessby 5, min, resulting in either severe (28%) or nor-BNI (35 and 50 nmol) improved survival rate (Fig. 7, upper moderate (22%) paralysis or death (50%) by 24 hr (Figs. 6, 7). panel). Nalmefene or nor-BNI limited these effects: each compound significantly improved hindlimb function measuredby neuro- Discussion score(Fig. 6), walking ability (Fig. 7, middle panel), and angle- Dyn A caused sustainedincreases in FFAs, as well as early board score (Fig. 7, lower panel), as compared to vehicle-pre- declinesin tissue phospholipid and cholesterol content. These treated controls. Effects of nalmefene were stereospecific: changeslikely reflect membranelipid hydrolysis, which hasbeen (-)nalmefene, but not (+)nalmefene, showed significant pro- well describedafter TSCI (Demediuk et al., 1985; Faden et al., tection. Actions of nor-BNI were doserelated: 20 and 35 nmol 1987). Phospholipid decomposition results in releaseof FFAs significantly improved neuroscoresat both 60 min and 24 hr, (Rehncrona et al., 1982) and is found after a variety of insults, whereas the 50-nmol dose did not (Fig. 6); the 2 lower doses including ischemia(Yoshida et al., 1982), hypoxia (Gardiner et
50 150 160 ASP GLU I GLY
40 120
80 Figure 5. Total rat spinal cord tissue levels of aspartate (ASP), glutamate 20 (GLU), and glycine (GLY) at the infu- sion site after intrathecal administra- tion of vehicle [saline + saline (veh)], saline + Dyn A (dyn), or nalmefene + l( Dyn A (nut) at 24 hr postinfusion. Val- ues are expressed as means+ SEM (n = 7 per group). a, differentfrom veh (p < 0.05); *, different from dyn (p < 0.05). C aa, different from veh (p < 0.005). veh dyn nal veh dw nal veh dyn nal The Journal of Neuroscience, December 1990, fO(12) 3797
Table 1. Effects of nalmefene pretreatment on outcome after intrathecal administration of Dyn Aa
60 min 24 hr Vehicle Nalmefene Vehicle Nalmefene Median neuroscore 1 5* 2 6* Percentwalkers 0 86* 40 100* Angle-board score 48 + 4 13 f 5* 21+3 7 f 2* 20 35 50 (4 (+I Survival rate 41.7% loo%* vehicle nor-BNI nalmefene a Data represent behavioral scoresand survival rates from animals killed at 24 hr for analysis of lipids or amino acids in Study 1.
*b -C0.05 compared to vehicle. 60
2%!i 60
9 40 al., 198 l), hypoglycemia (Agardh et al., 198l), and trauma (Fa- s den et al., 1987). FFAs may causecellular damageeither directly 20 or through the actions of their metabolites (Chan et al., 1983; Hsu et al., 1985). High concentrations of FFAs may directly 60 min 24h serve as detergents that disrupt cell membranes (Lucy, 1970), facilitate osmotic hemolysis (Raz and Livine, 1973), inhibit 50 synaptosomal uptake of amino acids, reduce synaptosomal Na+,K+-ATPaseactivity (Rhoadset al., 1982; Faden et al., 1987), F and impair mitochondrial function (Lazarewicz et al., 1972; u 30 Wojtczak, 1976). In addition, FFAs may contribute to free- 8 radical-induced tissue injury, believed to be important in the 2 20 0 pathophysiology of TSCI (Hall and Wolf, 1986). The decrease a, in cholesterolmay alsoreflect Dyn A-induced membranebreak- Fi 10 down. Cholesterol is known to have a condensingeffect on the 1 400 I acyl-chain region of fluid bilayers, contributing to structural 60 min 24 h stability; a decreasein cholesterol affects the permeability of lipid bilayers to water and ions suchas K+, Na+, and Ca2+(Blok 0 vehicle FJ nor-BNI (4 nalmefene H (+) nalmefene et al., 1977; HouSlay and Stanley, 1982). Spinal cord trauma causesa decline in tissue cholesterol (Demopoulos et al., 1982; Figure 7. In animalsfrom Figure 6, nor-BNI and nalmefenesignifi- cantly improvedcorresponding effects of Dyn A, including24-hr sur- Segler-Stahl et al., 1985), as well as changesin tissue cations vival rate (upper panel), functionalability (middle panel), and angle- (Lemke et al., 1987; Kwo et al., 1989). board score(lower panel, mean +- SEM). Asterisks, different from Administration of Dyn A causedsustained (at 24 hr) decreases vehicle-pretreatedcontrols (*, p < 0.05; **, p < 0.005). in total tissueglutamate, aspartate,and glycine, suggestingearly releaseof theseamino acids into the extracellular spacefollowed by transport into the circulation or metabolic transformation
tt tt t tt tt 7 ...... 1 t
Figure 6. Comparisonof effect of in- trathecal pretreatmentwith nor-BNI (dosesfrom left to right: 20, 35, 50 nmol),nalmefene (16 nmol),or vehicle on rat hindlimbneuroscore (at 60 min and 24 hr) after infusionof 24 nmol Dyn A. Each treatment significantly imoroved neuroscorescomnared to I 60 min 24 h coGrols (t, p < 0.05; j-f, p 2 O.OOS), with protective effects of nalmefene specific for the (-) stereoisomer. His- tograms represent median scores; dots indicate individual animal neuro- 0 vehicle q nor-BNI (-) nalmefene H (+) nalmefene scores. 3798 Bakshi et al. * Dynorphan-Induced Neurochemical Changes
(Erecinska et al., 1984). Spinal cord trauma causes sustained Yaksh, 1986; Long et al., 1988). Long et al. (1989b), using an decreases in total tissue levels of glutamate and aspartate (De- anesthetized rat model involving intrathecal injection by lumbar mediuk et al., 1989), as well as early transitory increases in puncture, failed to block Dyn A-induced paralysis with nor- extracellular concentrations of these amino acids (Panter et al., BNI; however, only a single, relatively low dose of nor-BNI was
1990). Preliminary studies from our laboratory utilizing in vivo evaluated. Because the K-agOniSt U50488H, even at high doses, microdialysis indicate that Dyn A administration to the rat does not cause alterations in motor function, some have argued spinal cord causes early extracellular increases of glutamate, that Dyn A-induced paralysis is entirely nonopioid (Stevens and aspartate, and glycine (A. Faden and P. Halt, unpublished ob- Yaksh, 1986; Long et al., 1988). servations). Glutamate and aspartate have a neurotoxic action Nalmefene is an opiate-receptor antagonist with increased that has long been recognized (Lucas and Newhouse, 1957; Ol- activity at K-Opiate receptors (Michel et al., 1985), which is ney et al., 197 1). Among the known EAA receptors, the NMDA strongly neuroprotective after spinal cord trauma (Faden et al., receptor appears to mediate this “excitotoxic” action (Olney et 1988). Nalmefene pretreatment attenuated the lipid and amino al., 197 1; Choi et al., 1988). Treatment with NMDA antagonists acid changes after Dyn A infusion and stereospecifically limited limits tissue damage after spinal cord trauma (Faden et al., the paralytic effects. In addition, the highly specific K-Opiate 1990). Extracellular increases in glycine may also contribute to receptor antagonist nor-BNI (Takemori et al., 1988) which lim- excitotoxicity: studies in vitro (Johnson and Ascher, 1987) and its paralysis after spinal cord trauma (Faden, 1990) reduced in vivo (Danysz et al., 1989) have shown that glycine potentiates Dyn A-induced motor dysfunction. Taken together, these find- glutamate-induced and NMDA-induced responses by an allo- ings strongly suggest that the consequences of Dyn A admin- steric binding site located on the NMDA receptor complex. The istration are, at least in part, mediated by opiate receptors, and hypothesis that extracellular increases in EAA and glycine con- more specifically, the K-Opiate receptor. Consistent with this tribute to Dyn A-induced irreversible paralysis and histological hypothesis, Dyn A binds well to opiate receptors, with prefer- damage is consistent with studies showing that NMDA receptor ence for K subtypes (Chavkin et al., 1982); such receptors are antagonists limit Dyn A-induced neurological dysfunction, in- widely distributed in the spinal cord (Traynor et al., 1982; cluding those acting at the glycine binding site (Bakshi and Fa- Czlonkowski et al., 1983; Mack et al., 1984; Gouarderes et al., den, 1990b), glutamate binding site (Caudle and Isaac, 1988; 1985). There is also mounting evidence to support the existence Bakshi and Faden, 1990a), and ion channel (Long et al., 1989a; of separate populations of K-receptOrS (isoreceptors) within the Bakshi and Faden, 1990a). CNS (Attali et al., 1982; Gouarderes and Cros, 1984; Iyengar The mechanism by which dynorphin induces NMDA-recep- et al., 1985; Neck et al., 1988; Zukin et al., 1988); therefore, tor-mediated actions remains speculative. Dyn A causes marked Dyn A and U50488H may well be acting through different iso- reduction in spinal cord blood how upon intrathecal adminis- receptors. At least 2 subpopulations of K-receptors have been tration (Long et al., 1987; Thornhill et al., 1989). Thus, Dyn identified in the rat spinal cord: high-affinity (K,) and low-affinity A-induced ischemia may trigger the release of EAAs, leading to (K,) sites (Gouarderes and Cros, 1984). Similarly, Zukin et al. biomembrane breakdown and the liberation of FFAs, activation (1988) have identified KI and K2 sites in the rat brain; though of NMDA receptors, and cell death with subsequent functional Dyn A and U50488H show similar potencies at K,, Dyn A is deficits. It is also possible that Dyn A causes release of EAAs nearly 300 times more active then U50488H at the low-affinity through opiate-receptor mechanisms independent of ischemia, K2 Site. possibly via presynaptic actions. However, the relationship be- Our findings may have particular application to spinal cord tween dynorphin and NMDA-receptor-mediated activity is or brain trauma, which cause the accumulation of immuno- complicated, with in vitro evidence that dynorphin may serve reactive Dyn A (Faden et al., 1985; McIntosh et al., 1987) and through “nonopioid” actions as an NMDA antagonist (Choi et a decrease in blood flow (Young et al., 1981; McIntosh et al., al., 1989; Massardier and Hunt, 1989). 1987), as well as the release of FFAs (Faden et al., 1987) and Involvement of opiate receptors in the neurologic effects of EAAs (Faden et al., 1989; Panter et al., 1990). Neurological Dyn A was first suggested by Han and Xie (1982), who dem- dysfunction after such injuries can be limited by treatment with onstrated that naloxone partially blocks the tail-flick effects of Dyn A antiserum (Faden, 1990), NMDA antagonists (Faden Dyn A-( l-l 3); the low efficacy of naloxone to reverse these and Simon, 1988) or opiate antagonists (Faden et al., 1988). effects and the absence of cross-tolerance between morphine The similar neurochemical, physiological, and pharmacological analgesia and Dyn A-induced reflex loss led them to propose profiles between dynorphin-induced and traumatic spinal cord that Dyn A was acting not at p-receptors, but at K-receptors. injuries strengthen the hypothesis that endogenous dynorphin Subsequently, a number of groups have demonstrated that opioid is a secondary injury factor, contributing to the pathogenesis of antagonists attenuate Dyn A-induced neurologic dysfunction, CNS trauma. These studies also suggest that dynorphin-induced including the nonselective antagonist naloxone (Przewlocki et tissue damage may result, in part, from the release of EAAs and al., 1983; Spampinato and Candeletti, 1985), the K-active an- phospholipid hydrolysis, providing a potential linkage among tagonist MR1452 (Spampinato and Candeletti, 1985), and the opioid, excitotoxin, and membrane lipid mechanisms of sec- K-Selective antagonist nor-BNI (Faden, 1990). In contrast, other ondary injury after neurotrauma. observations suggest that the effects of Dyn A are not entirely opioid. Dyn A fragments (2-l 7) and (3-13) apparently devoid References of opiate-receptor binding activity (Chavkin and Goldstein, 198 1; Agardh CD, Chapman AG, Nilsson B, Siesjo BK (198 1) Endogenous Walker et al., 1982), also cause a clinically similar paralysis, substrates utilized by rat brain in severe insulin-induced hypoglyce- though with less potency (Faden and Jacobs, 1984; Stevens and mia. J Neurochem 36:490-500. Allen KG, Fellows ME, Tomheim PA, Wagner KR (1984) A new Yaksh, 1986; Long et al., 1988; Bakshi and Faden, 1990a). procedure to analyze free fatty acids. Application to 20-mg brain tissue Furthermore, some groups have failed to block Dyn A-induced samples. J Chromatogr 309:33-42. paralysis with naloxone (Faden and Jacobs, 1984; Stevens and Allison LA, Mayer GS, Shoup RE (1984) o-Phthalaldehyde derivatives The Journal of Neuroscience, December 1990, fO(12) 3799
of amines for high speed liquid chromatography/electrochemistry. Faden AI, Sacksen I, Noble LJ (1988) Opiate receptor antagonist Anal Chem 56:1089-1096. nalmefene improves neurological recovery after traumatic spinal cord Arias MJ (1985) Effect of naloxone on functional recovery after ex- injury in rats through a central mechanism. J Pharmacol Exp Ther perimental spinal cord injury in the rat. Surg Neurol23:440-442. 245~742-748. Attali B, Gouarderes C, Mazurguil H, Audigier Y, Cros J (1982) Ev- Faden AI, Demediuk P, Panter SS, Vink R (1989) Role of excitatory idence for multiple “kappa” binding sites by use of opioid peptides amino acids and NMDA receptors in traumatic brain injury. Science in the guinea-pig lumbosacral spinal cord. Neuropeptides 4:53-64. 244:798-800. Bakshi R. Faden AI (1990a) Comnetitive and non-comnetitive NMDA Faden AI, Ellison JA, Noble LJ (1990) Effects of competitive and non- antagonists limit dynorphin A-induced rat hindlimb paralysis. Brain competitive NMDA receptor antagonists in spinal cord injury. Eur J Res 507: l-5. Pharmacol 175:165-174. Bakshi R, Faden AI (1990b) Blockade of the glycine modulatory site Flamm ES, Young W, Demopoulos HB, DeCrescito V, Tomasula JJ of NMDA receptors modified dynorphin-induced behavioral effects. (1982) Experimental spinal cord injury: treatment with naloxone. NeurosciLett 110:113-117. Neurosurgery 10:227-23 1. Blok MC, Van Deenen LLM, De Gier J ( 1977) The effect of cholesterol Gardiner M, Nilsson B, Rehncrona S, Siesjo BK (198 1) Free fatty incorporation on the temperature dependence of water permeation acids in moderate and severe hypoxia. J Neurochem 36: 1500-l 505. through liposomal membranes prepared from phosphatidylcholines. Gouarderes C, Cros J (1984) Opioid binding sites in different levels Biochim Biophys Acta 464:509-5 18. of rat spinal cord. Neuropeptides 5: 113-l 16. Bowman RE, Wolf RC (1962) A rapid ultramicro method for total Gouarderes C, Cros J, Quiron R (1985) Autoradiographic localization serum cholesterol. Clin Chem 8:302-309. of mu, delta and kappa opioid receptor binding sites in rat and guinea Bradford M (1976) A rapid and sensitive method for the quantitation pig spinal cord. Neuropeptides 6:331-342. - of microgram quantities of protein utilizing the principle of dye bind- Hall ED. Wolf DL (1986) A nharmacoloaical analvsis of the natho- ing. Anal Biochem 72:248-254. physiological mechanisms of-post-traumatic spinal cord ischemia. J Caudle RM, Isaac L (1987) Intrathecal dynorphin (1 - 13) results in an Neurosurg 64:95 l-96 1. irreversible loss of the tail flick reflex in rats. Brain Res 435:1-6. Han J-S, Xie C-W (1982) Dynorphin: potent analgesic effect in spinal Caudle RM, Isaac L (1988) A novel interaction between dynorphin cord ofthe rat. Life Sci 31:1781-1784. (1-13) and an N-methyl-D-aspartate site. Brain Res 443:329-332. Hara A, Radin NS ( 1978) Lipid extraction of tissues with a low toxicity Chan PH, Fishman RA, Carronna J, Schmidley JW, Piroleau G, Lee J solvent. Anal Biochem 90:420-426. (1983) Induction of brain edema following intracerebral injection of Hayes RL, Galinet BJ, Kulkarne P, Becker D (1983) Effects of nal- arachidonic acid. Ann Neurol 13~625-632. oxone on systemic and cerebral responses to experimental concussive Chavkin C. Goldstein A (198 1) Snecific receutor for the ouioid peutide brain injury in cats. J Neurosurg 58:720-728. dynorphin: structure-activity relationships: Proc Nat1 Acad S‘ci -USA Hayes RL, Jenkins LW, Lyeth BG (1988) Pretreatment with phen- 78~6543-6547. cyclidine, an N-methyl-D-aspartate receptor antagonist, attenuates long- Chavkin C, James IF, Goldstein A (1982) Dynorphin is a specific term behavioral deficits in the rat produced by traumatic brain injury. endogenous ligand of the K-opioid receptor. Science 2 15:4 13-4 15. J Neurotrauma 5:259-274. Choi DW, Koh J, Peters S (1988) Pharmacology of glutamate neu- Herman BH, Goldstein A (1985) Antinociception and paralysis in- rotoxicity in cortical cell culture: attenuation by NMDA antagonists. duced by intrathecal dynorphin A. J Pharmacol Exp Ther 232:27- J Neurosci 8:185. 32. Choi DW, Rose K, Weiss JH, Faden AI (1989) Dynorphin attenuates Houslay MD, Stanley KK (1982) Dynamics of biological membranes. NMDA receptor-mediated cortical neuronal injury in vitro. Sot Neu- Influence on synthesis, structure and function, pp 7 l-8 1. New York: rosciAbstr 15:1113. Wiley. Czlonkowski A, Costa T, Przewlocki R, Pasi A, Herz A (1983) Opiate Hsu CY, Halushka PV, Hogan EL, Banik NL, Lee WA, Perot LP (1985) receptor binding sites in human spinal cord. Brain Res 267~392-396. Alteration of thromboxane and prostacyclin levels in experimental Danysz W, Wroblewski JT, Brooker G, Costa E (1989) Modulation sninal cord iniurv. Neuroloav 35: 1003-1009. of glutamate receptors by phencyclidine and glycine in the rat cere- Inoue Y (1986) Evoked spinal potentials in the Wistar rat: effect of belhtm: cGMP increases in viva..Brain Res 479:270-276. cord compression and drugs. J Japan Orthop Assoc 60:777-785. Demediuk P. Saunders RD. Anderson DK. Means ED. Horrocks LA Iyengar S, Kim H, Wood PL (1985) In vivo evidence for kappa iso- (1985) Membrane lipid changes in laminkctomized and traumatized receptors in rat: neurochemical and neuroendocrine indices. J Neu- cat spinal cord. Proc-Nat1 Acad Sci USA 82:707 l-7075. rochem 44:S89. Demediuk P, Daly MP, Faden AI (1989) Effect of impact trauma on neurotransmitter and non-neurotransmitter amino acids in rat spinal Johnson JW, Ascher P (1987) Glycine potentiates the NMDA response cord. J Neurochem 52:1529-1536. in cultured mouse brain neurons. Nature 325:529-53 1. Demonoulos HB. Flamm ES. Selieman ML. Pietroniaro DD. Tomasula Jones J, Keenan RW, Horowitz P (1982) Use of 6-p-toluidine-2-naph- J, DkCrescito V (1982) l&h& studies on free-ridical pathology in thalenesulfonic acid to quantitate lipids after thin layer chromatog- the major nervous system disorders: effect of very high doses of meth- raphy. J Chromatogr 237~522-524. ylprednisolone on the functional outcome, morphology, and chem- Kwo S, Young W, DeCrescito V (1989) Spinal cord sodium, potas- istry of experimental spinal cord impact injury. Can J Physiol Phar- sium, calcium and water concentration changes in rats after graded macol60:1415-1424. contusion injury. J Neurotrauma 6: 13-24. Erecinska M, Nelson D, Wilson DF, Silver IA (1984) Neurotransmitter Lazarewicz JW, Strosznajder J, Gromek A (1972) Effects of ischemia amino acids in the CNS. I. Regional changes in amino acid levels in and exogenous fatty acids on the energy metabolism in brain mito- rat brain during ischemia and reperfusion. Brain Res 304~9-22. chondria. Bull Acad Pol Sci [Biol] 203599-606. Faden AI (1990) Opioid and non-opioid mechanisms may contribute Lemke M, Demediuk P, McIntosh TK, Vink R, Faden AI (1987) Al- to dynorphin’s pathophysiologic actions in spinal cord injury. Ann terations in tissue Mg”, Na+ and spinal cord edema following impact Neurol 27167-74. trauma in rats. Biochem Biophys Res Commun 147:1170-l 175. Faden AI, Jacobs TP (1984) Dynorphin-related peptides cause motor Lemke M, Yum SW, Faden AI (1990) Lipid alterations correlate with dysfunction in the rat through a non-opiate action. Br J Pharmacol tissue magnesium decrease following impact trauma in rabbit spinal 8 1:271-276. cord. Mol Chem Neuropath, in press. Faden AI, Simon RP (1988) A potential role for excitotoxins in the Long JB, Kinney RC, Malcolm DS, Graeber GM, Holaday JW (1987) pathophysiology of spinal cord injury. Ann Neurol 231623-626. Intrathecal dynorphin A (l- 13) and dynorphin A (3- 13) reduce rat Faden AI, Jacobs TP, Holaday JW (198 1) Opiate antagonist improves spinal cord blood flow by nonopioid mechanisms. Brain Res 436: neurologic recovery after spinal injury. Science 211:493-494. 374-379. Faden AI, Molineaux CJ, Rosenberger JG, Jacobs TP, Cox BM (1985) Long JB, Petras JM, Mobley WC, Holaday JW (1988) Neurological Increased dynorphin immunoreactivity in spinal cord after traumatic dysfunction after injection of dynorphin A-( 1- 13) in the rat. II. Non- injury. Reg Pept 11:3541. opioid mechanisms mediate loss of motor; sensory and autonomic Faden AI, Chan PH, Longar S (1987) Alterations in lipid metabolism, function. J Pharmacol EXD Ther 246: 1167-l 174. Na+,K+-ATPase activity, and tissue water content of spinal cord fol- Long JB, Rigamonti DD, Martinez-Arizala A, Holaday JW (1989a) lowing experimental traumatic injury. J Neurochem 48: 1809-l 8 16. Non-competitive iV-methyl-D-aspartic acid receptor inhibitors pre- 3800 Bakshi et al. l Dynorphan-Induced Neurochemical Changes
vent persistent dynorphin A-induced hindlimb paralysis in rats. J Rouser G, Siakotos V, Fleischer S (1969) Quantitative analysis of Neurotrauma 659-60. phospholipids by thin layer chromatography and phosphorus analysis Long JB, Rigamonti DD, de Costa B, Rice KC, Martinez-Arizala A of spats. Lipids 1:85-86. ( 1989b) Dynorphin A-induced rat hindlimb paralysis and spinal cord Saunders RD, Horrocks LA (1984) Simultaneous extraction and prep- injury are not altered by the K-opioid antagonist nor-binaltorphimine. aration for HPLC of prostaglandins and phospholipids. Anal Biochem Brain Res 497~155-162. 143:71-75. Lucas DR, Newhouse JP (1957) The toxic effect of sodium L-glutamate Segler-Stahl K, Demediuk P, Castillo R, Watts C, Moscatelli EA (1985) on the inner layers of the retina. Arch Ophthalmol 58: 193-20 1. Phospholipids of normal and experimentally injured spinal cord of Lucy JA (1970) The fusion of biological membranes. Nature 227:8 15- the miniature pig. Neurochem Res 10:563-569. 817. Spampinato S, Candeletti SU (1985) Characterization of dynorphin Mack KJ, Killian A, Weyhanmeyer JA (1984) Comparison of mu, A-induced antinociception at the spinal level. Eur J Pharmacol 110: delta, and kappa opiate binding sites in rat brain and spinal cord. 21-30. Life Sci 34:281-285. Stevens CW, Yaksh TL (1986) Dynorphin A and related peptides Massardier D, Hunt PF (1989) A direct non-opiate interaction of administered intrathecally in the rat: a search for putative kappa dynorphin-( 1- 13) with the iV-methyl-D-aspartate (NMDA) receptor. opiate receptor activity. J Pharmacol Exp Ther 238:833-838. Eur J Pharmacol 170:125-126. Takemori AE, Ho BY, Naesbeth JS, Portoghese PS (1988) Nor-bin- McIntosh TK, Hayes RL, Dewitt DS, Agura V, Faden AI (1987) altorphimine, a highly selective K-opioid antagonist in analgesic and Endogenous opioids may mediate secondary damage after experi- receptor binding assays. J Pharmacol Exp Ther 246:255-258. mental brain injury. Am J Physiol 253:E565-E574. Thornhill JA. Gregor L. Mathison R. Pittman 0 (1989) Intrathecal Michel ME, Bolger G, Weissman B-A (1985) Binding of a new opiate dynorphin ‘A administration causes’ pressor responses in rats associ- antagonist, nalmefene, to rat brain membranes. Meth Find Exp Clin ated with an increased resistance to spinal cord blood flow. Brain Res Pharmacol 7: 175-l 77. 490:174-177. Neck B, Rajpara A, O’Connor LH, Cicero TJ (1988) Autoradiography Traynor JR, Kelley PD, Rance MJ (1982) Multiple opiate binding of PHlU-69593 binding sites in rat brain: evidence for K opioid re- sites in rat spinal cord. Life Sci 3 1: 1377-l 380. ceptor-subtypes. Eur J Pharmacol 154:27-34. Walker JM, Moises HC, Coy DH, Bald&hi G, Akil H (1982) Non- Olney JW, Ho OL, Rhee V (197 1) Cytotoxic effects of acidic and opiate effects of dynorphin and des-Tyr-dynorphin. Science 2 18: 1136- sulfur containing amino acids on the infant mouse central nervous 1138. system. Exp Brain Res 14:61-76. Wojtczak L (1976) Effect of long-chain fatty acids and acyl-CoA on Panter SS. Yum SW. Faden AI (1990) Alterations in extracellular mitochondrial permeability, transport and energy-coupling processes. amino acids after traumatic spinal cord injury. Ann Neurol 27:96- J Bioenerg Biomembr 8:293-3 11. 99. Yaksh TL, kudy TA (1976) Chronic catheterization of the spinal Przewlocki R, Shearman GT, Hen A (1983) Mixed opioidinon-opioid subarachnoid snace. Phvsiol Behav 17: 103 l-1036. effects of dynorphin and dynorphin-related peptides after their in- Yoshida S, Abe K, Bustd R, Watson BO, Kogure K, Ginsberg MD trathecal injection in rats. Neuropeptides 3:233-240. (1982) Influence of transient ischemia on lipid-soluble antioxidants, Raz A, Livine A (1973) Differential effects of lipids on the osmotic free fatty acids and energy metabolites in rat brain. Brain Res 245: fragility of erythrocytes. Biochim Biophys Acta 311:222-229. 307-316. Rehncrona S, Westerberg E, Akesson B, Siesjo BK (1982) Brain cor- Yoshimura K, Huidobro-Toro JP, Lee NM, Loh HH, Way EL (1982) tical fatty acids and phospholipids during and following complete and Kappa-opioid properties of dynorphin and its peptide fragments on severe incomplete ischemia. J Neurochem 38:84-93. the guinea pig ileum. J Pharmacol Exp Ther 222:7 l-79. Rhoads DE, Kaplan MA, Peterson NA, Raghupathy E (1982) Effects Young W, Flamm ES, Demopoulos HB, Tomasula JJ, DeCrescito V of free fatty acids on synaptosomal amino acid uptake systems. J (198 1) Naloxone ameliorates posttraumatic ischemia in experimen- Neurochem 38:1255-1260. tal spinal contusion. J Neurosurg 55:209-219. Rice KC, Newman AH (1986) Total synthesis of northebaine, nor- Zukin RS, Eghbali M, Olive D, Unterwald EM, Tempel A (1988) morphine, noroxymorphone enantiomers and derivatives via N-nor- Characterization and visualization of rat and guinea pig brain K-opiate intermediates. Application for U.S. Patent (filed 10/3 l/86). receptors: evidence for K~and K>opioid receptors. Proc Nat1 Acad Sci Rivlin AS, Tator &I (1977) Objective clinical assessment of motor USA 85:40614065. function after experimental spinal cord injury in the rat. J Neurosurg 47:577-581.